1. Field of the Invention
The present invention relates to a scanning microscope used as a research tool for a biological specimen and to a specimen image obtaining method.
2. Description of the Related Art
Heretofore, a scanning laser microscope has been known as a scanning microscope used as a research tool for a biological specimen wherein laser light is converged on a point on a specimen, and this convergence point is scanned in a two-dimensional direction on the specimen to obtain two-dimensional luminance information on the specimen, for example, as disclosed in Jpn. Pat. Appln. KOKAI Publication No. 10-10436.
Laser light comprising a single beam is used for the scanning laser microscope, and since the laser light has a good convergence property, it is effective in obtaining optical information on a minute point on an XY plane of the specimen. Moreover, because detection light from the specimen is detected by a confocal optical system that detects light through a pinhole located at a position optically conjugate with the convergence point in the specimen, it is possible to eliminate light from out-of-focus positions, and more accurate optical information can be obtained.
Therefore, according to a confocal laser microscope, it is possible to obtain optical information on a point in a three-dimensional space of the specimen. Further, laser light is scanned along the XY plane, an XZ plane, and other two-dimensional planes, and the above-mentioned information for each point can be arranged in accordance with scanned positions, so that optical slice images can be formed. Moreover, a galvanometer mirror is generally used to scan a laser beam. Two galvanometer mirrors are preferably combined so as to scan in XY directions, respectively, so that an imaging region is XY-scanned for each line as a raster scan of a television. In the raster scan, the laser light is sequentially applied to points adjacent in an X direction, and light (fluorescence, reflected light, etc.) from the specimen obtained at that moment is detected by a detector. In this case, a time difference from detection of the fluorescence after application of laser light to arrival at a next point (Xn+1, Yn+1) is in an order of microsecond.
Meanwhile, the confocal laser microscope is effective in a caged method and the like used for fluorescence observation. Here, the caged method is an observation method wherein a caged indicator and a fluorescence indicator that is sensitive to a calcium ion concentration are injected into a specimen, and stimulating laser light is applied to a certain portion of the specimen, so that a caged radical of the caged indicator is cleaved, substances contained therein are released, and a change over time in the calcium ion concentration at that point is fluorescence-observed by application of observation excitation laser light to the specimen. According to this method, for example, when a caged compound to which calcium ions are bonded is introduced into the specimen, application of UV laser for release of the caged compound at a point (Xn, Yn) causes the caged compound located at this position to be cleaved, and Ca2+ ions retained therein are released. The observed specimen causes a certain reaction to the calcium ions. Fluorescence generated by this reaction is detected by a detector to observe how the specimen reacts to a stimulus of the calcium ions.